Method for testing blood units for viral contamination

ABSTRACT

The present invention provides a method for testing a blood unit for viral contamination without rendering the blood unit unusable for therapeutic applications. The method comprises the steps of: removing and collecting from a blood unit a majority of the leukocytes present therein with a leukocyte filter; and using the leukocytes collected in and on the filter to test the blood unit for viral contamination. The present invention also provides a method for validating viral inactivation processes.

This application is a continuation of application Ser. No. 07/833,285,filed Feb. 10, 1992 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to diagnostic techniques. Morespecifically, the present invention relates to the testing of bloodunits for viral contamination.

In a variety of therapies, such as transfusion and transplants, bodyfluids, especially blood components, such as red blood cells, platelets,plasma, and bone marrow, are infused from one or more individuals into apatient. Although such therapies provide treatments, some of which arelife saving, that cannot otherwise be provided, due to the transmissionof infectious diseases there may be potential risks to such therapies.

For example, it is known that blood can carry infectious agents, such ashepatitis virus, human immunodeficiency virus (an etiological agent forAIDS), cytomegalovirus, Epstein Barr virus, and herpes virus. Althoughscreening methods exist to identify blood that may include such viruses,current screening methods do not assure that every blood unit thatcontains such a virus is identified.

In this regard, one of the difficulties in testing blood components forviral contamination, such as HIV, is that many current diagnostic testsare based on an identification of antibodies. Accordingly, they willonly exhibit a positive test result if the blood unit includesantibodies for the virus, e.g., anti-HIV. With respect to intracellularviral infections, an individual, however, does not generate antibodiesimmediately upon infection. Rather there is a "window period" thatextends from the initial infection of the patient with a virus to thegeneration of antibodies. When an individual is in this window period,diagnostic tests that are based on antibodies will not identify theindividual, or the blood unit, as being infected. But, even thoughantibodies are not present, the blood unit can still transmit aninfection.

It is believed that this window period, with respect to HIV infection,extends from approximately six weeks to 48 months. During this timeperiod, an individual who has been infected with HIV and accordingly,whose blood will transmit same, will register a negative antibodyresponse. Therefore, current screening methods will not identify asviral contaminated a blood unit from an individual who is infected withHIV but who has not generated anti-HIV.

In order to identify blood units that may be contaminated because anindividual is within the window period, recent attempts have focussed onthe use of nucleic acid sequencing diagnostic techniques. Specifically,attempts have been made to use polymerase chain reaction (PCR)techniques for detecting nucleotide sequences for HIV virus.

A number of methods of using PCR are known. Briefly, in one PCR method,a sample containing DNA is placed in a reaction tube includingappropriate buffers, nucleoside triphosphates, a thermostable DNApolymerase and oligonucleotide primers complementary to the ends of aregion of DNA of interest. Initially, to denature the double-strandedDNA under study, the temperature of the reaction is rapidly increased.The temperature is then decreased allowing the oligonucleotide primersto anneal to their complementary sequences. By increasing thetemperature, a DNA extension occurs at the optimal temperature ofactivity for the polymerase. By repeating these cycles ofdenaturation-annealing-extension, a single sequence of a few hundredbase pairs can be amplified. This amplification can be in the range of afactor of 10⁶ and detected with relative ease. Conway, "Detection ofHIV-1 by PCR in Clinical Specimens", "Techniques in HIV Research"Stockton Press (1990)

Although nucleic acid sequencing techniques are very sensitive, they arealso sample specific. Moreover, the samples of the blood units so testedare rendered unusable for therapeutic applications. Because of this,currently nucleic acid sequencing diagnostic methods do not provide amethod for insuring that blood units do not have viral contamination.

For example, if one were to attempt to use a PCR technique to test asample of a blood unit using standard technology, one could not insurethat the unit did not contain viral contamination even if the test wasnegative. In this regard, because a PCR test is sample specific unlessthe whole blood unit was tested, it would not accurately reflect thatthe unit did not include viral contamination; if only a 10 ml samplewere tested, the remaining 290 ml of a 300 ml could contain a viralcontaminant.

A PCR test will only determine if there is a viral agent in the samplebeing assayed, not in the entire unit. Because the test destroys theviability of the component, heretofore, it was not possible to test theentire component to ensure that it is not contaminated with, forexample, HIV.

Accordingly, there is a need for improved diagnostic testing of bloodunits to determine viral contamination during the "window period" ofcontamination.

SUMMARY OF THE INVENTION

The present invention provides a method for testing a blood unit forviral contamination that does not render the blood unit unusable fortherapeutic applications. The method allows one to effectively samplethe entire blood unit using, for example, nucleic acid sequencingtechniques. The method improves the accuracy of diagnostic tests forviral contaminants.

Although typically to increase the accuracy of a diagnostic test forinfectious disease it is desirable to increase the sample volume of thematerial being assayed, with respect to blood components this is notpossible. In the case of blood and its components, only a small fractionof the total volume of the blood component can be sacrificed forperformance of diagnostic assays for infectious diseases. The inabilityto sample the whole blood unit places a finite limit on the accuracy ofdiagnostic testing (including nucleic acid amplification technology) dueto sampling volume. In this regard: 1) the assay result can be a truenegative, but the unit positive; or 2) the assay result can be a falsenegative due to lack of sensitivity of the assay. The present inventionaddresses both of these limitations.

To this end, the present invention provides a method for testing a bloodunit for viral contamination without rendering the blood unit unusablefor therapeutic applications. The method comprises the steps of:removing and collecting from a blood unit a majority of the leukocytespresent therein; and using the collected leukocytes to test the bloodunit for viral contamination.

In an embodiment, the present invention provides a method for testing ablood unit for viral contamination without rendering the blood unitunusable for therapeutic applications comprising the steps of: passing ablood component through a leukocyte filter to remove a majority of theleukocytes contained within the blood component; collecting a resultantfiltered blood component; creating a cell lysate by passing a lysingreagent through the leukocyte filter; and assaying the cell lysate forviral contaminants.

In an embodiment, the method includes the step of culturing the filterbefore passing a lysing reagent through the filter. The filter can becultured for 1 to 5 days.

In an embodiment, the present invention provides a method for testing ablood unit for viral contamination without rendering the blood unitunusable for therapeutic applications comprising the steps of separatingthe blood unit into a plasma rich platelet component, a buffy layer, anda red blood cell component; removing the buffy layer; and assaying thebuffy layer for viral contamination.

The present invention also provides a process that can be used invalidation studies for viral inactivation or removal processes.

To this end, a method for the validation of a viral inactivation processis provided comprising the steps of: adding a virus to a blood component(or in the alternative, using a naturally contaminated blood component);treating the blood component with a viral inactivation process; addingvirus host cells to the blood component; filtering the blood componentto remove the virus host cells; and testing the virus host cells forviral contaminants.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the detailed description of thepresently preferred embodiments and from the drawings.

FIELD DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically an embodiment of the method of thepresent invention for testing a blood component for viral contamination.

FIG. 2 illustrates schematically a further embodiment of the method ofthe present invention for testing a blood component for viralcontamination.

FIG. 3 illustrates schematically the use of an embodiment of the presentinvention for validating virus depletion/inactivation process.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The present invention provides an improved method for testing a bloodcomponent for a viral contaminant. The method of the present inventionimproves the accuracy of diagnostic tests without sacrificing the bloodunit to be administered. To this end, the process of the presentinvention allows the entire unit of whole blood, red blood cells, orplatelet concentrate to be sampled for viral contamination withoutrendering the cell product unusable for therapeutic applications.

In an embodiment, the invention includes the steps of using a filter tocapture leukocytes without damaging the blood components.

There are three principal indications for the transfusion of a bloodproduct: 1) deficiency in oxygen-carrying red blood cells; 2) deficiencyin hematologic factors related to blood clotting, includes platelets orprotein coagulation factors; and 3) deficiency in plasma volume.Patients requiring a transfusion do not receive whole blood but thespecific component required to overcome the clinical deficiency. Forexample, patients undergoing chemotherapy or radiation therapy requireprimarily platelets and to a lesser degree red cells. Bone marrow orother organ transplant and dialysis patients generally require only redblood cells.

Leukocytes are unwanted because they are not relevant to the therapeuticeffects of oxygen-carrying red blood cells, platelet or plasma, and havebeen implicated as increasing the risks associated with bloodtransfusion for their role in alloimmunization to HLA antigens and posttransfusion infection by acting as virus carrying cells. Typically, theleukocytes are removed by a filtering process in approximately 10% ofthe blood components that are transfused.

When a filtering process is used, the leukocytes captured in thesefilters are discarded. As set forth in detail below, pursuant to thepresent invention, the filters containing the leukocytes can be used asa starting material for a process to recover and/or amplify viralmarkers (vital nucleic acid sequences and/or antigens).

Pursuant to an embodiment of the present invention, a majority, greaterthan 50%, of the leukocytes are collected. Depending on the method used,up to 99.8% of the leukocytes may be collected. Leukocytes typicallycomprise 0.25% of the cellular composition of blood. A leukocyte countof 7.5 million/ml of blood is considered average for an adult. In a 500ml unit of whole blood, containing 63 ml of anticoagulant solution,3.2×10⁹ leukocyte cells will be present.

Because the leukocytes are a source of infectivity in the blood, theyprovide a desirable material for diagnostic assays. Furthermore, becausethe collected leukocytes are derived from the entire unit of bloodcomponent to be transfused, as opposed to a small segment thereof, animproved diagnostic procedure is achieved.

FIG. 1 illustrates schematically an embodiment of the process of thepresent invention. The blood component (which can be whole blood, redcell concentrate, platelet concentrate, or pooled platelet concentrate)is first passed through a filter which is designed to capture 90 togreater than 99% of the leukocytes present.

Leukocyte filters that can be used pursuant to the present inventioninclude the Sepacell R-500 filter available from Asahi Corp., Tokyo,Japan and the RC-100 for blood filtration and PL-100 for plateletfiltration, both available from Pall Biomedical Corp., East Hills, N.Y.It has been reported in Biotechnology & Medical, Aug. 19, 1988 that anAsahi filter is able to remove 99.8% of the leukocytes present in wholeblood.

The filtered blood component is collected in a blood collection bag. Theblood component is stored pending the results of the diagnostic test.

Pursuant to the present invention, the leukocyte filter is utilized as asource material for diagnostic tests for infectious disease antigen andnucleic acid sequences. To this end, the filter is treated to lyse thecells contained in the filter. This allows the recovery of nucleic acidsand antigens that are contained on and within the leukocytes.

The cells are lyse by using a lysing reagent. The lysing reagentpreferably will be a solution containing a detergent to break thecytoplasmic membrane and release the nuclei of the cells.

The cell lysate is then flushed out of the filter using an isotonicsaline solution and collected. This resultant cell lysate is then usedas a test material for diagnostic assays (nucleic acid amplificationtechnology for viral nucleic acid sequences, e.g., PCR 3SR or viralantigens, e.g., enzyme-linked immunoadsorbent assays).

Because the assay, pursuant to the present invention, will be based onan analysis of the great majority of the leukocytes present in the bloodunit, even when using sample specific analysis such as PCR methods onecan be assured that a negative result is accurate even if an individualis in a window period of infection.

Once a negative result is obtained using the cell lysate, the bloodcomponent contained in the collection container can then be transfusedinto a patient.

FIG. 2 illustrates schematically another embodiment of the presentinvention. If necessary to increase assay sensitivity (particularly forantigen capture assay systems), the leukocyte filter can be utilized asa culture chamber for viral replication.

In this case, again a blood component (whole blood, red cellconcentrate, platelet concentrate, or pooled platelet concentrate) isfiltered through a leukofilter. The leukofilter is separated from theblood bag container, containing the filtered blood component, and thefilter is flushed with tissue culture media such as 10% Fetal Calf Serumin RPMI 1640 media. In some cases, it may be desirable to supplement themedia with cytokines (e.g., IL-2 or T-cell mitogen (e.g., PHA)) topromote viral replication in the leukocytes and to sustain the viabilityof the cells in the filter.

In addition, in some cases, it may be desirable if the tissue culturemedia is supplemented with viral host cell lines (example H9 cells forHIV). In this case, the filter would be used as a culture chamber forthe leukocytes captured from the blood and for virus host cells. The H9cells can then be infected by HIV present in the captured leukocytes andcan serve to amplify the production of viral antigen (e.g., HIV p24antigen).

After the appropriate culture period of approximately 1 to 5 days, thefilter can be treated to accomplish lysis of the trapped cells by one ofseveral means (freeze-thaw, detergent, hypotonic shock). The resultingcell lysate can then be collected by flushing the filter with anisotonic saline solution.

The collected cell lysate can be tested using conventional antigencapture assay systems such as, for example, DuPont HIV p24 Care Profile,Elisa Kit or Coulter HIV p24 Antigen Assay (Hialeah, Florida) for theHIV p24 antigen or by nucleic acid amplification methodologies, such asGene Amplimer HIV-1 Reagents with GeneAmp® PCR Core Reagents andPerkin-Elmer Cetus DNA Thermal Cycler, available from Perkin-ElmerCetus. In some cases, the cell lysis may not be required due to releaseof the virus into the tissue culture media from infected cells.

Again, by using this method, even if a sample specific diagnostic testis used, because the test is with respect to the great majority ofleukocytes present, a negative test will ensure that the blood componentis not contaminated. The blood component contained in the blood bagcontainer can then be transfused into a patient.

In an embodiment of the method of the present invention, instead offiltering the blood component to capture the leukocytes, centrifugationprocesses that separate a whole blood unit into various components canbe used. During a typical centrifugation process, the whole blood isseparated in a top portion including platelet rich plasma, a middle orbuffy coat layer containing white cells and leukocytes, and a bottom redblood cell rich layer. Typically, the buffy coat layer is discarded.

Pursuant to the present invention, the buffy coat layer is used fordiagnostic applications. To this end, the buffy coat can be subjected tonucleic acid amplification, e.g., PCR or 3SR or viral antigen captureassay. By testing the buffy coat layer, if a negative test results, oneis assured that both the platelet rich plasma component and red bloodcell component are viral contaminant free. It is believed that bycollecting the buffy coat using standard techniques, at leastapproximately 80% of the leukocytes are recovered.

In an embodiment, the following method is used:

1. Centrifuge the blood bag in an inverted position in a refrigeratedcentrifuge containing whole blood at 5° C. using a "heavy" spin.

2. Hang the centrifuged, inverted bag on a ring stand or inverted plasmaexpressor. The temperature of the blood must not exceed 10° C. duringthe procedure. Allow the bag to hang undisturbed for several minutes.

3. Place the transfer bag on a scale (e.g., dietary scale) below theblood bag. Adjust the scale to zero.

4. Penetrate the closure of the primary bag, avoiding agitation of thecontents and allow red blood cells to flow to the transfer bag. At least80% of the red blood cells must be transferred to the satellite bag. Tocalculate the amount of red blood cells to be expressed estimate theamount of blood (excluding anticoagulant) in the bag multiplied by thedonor's hematocrit (assume 40% for females and 43% for males) (1 ml ofred blood cells (RBC) weighs 1.06 gm).

5. The remaining blood in the bag contains the buffy coat, some residualred blood cells, and the plasma.

6. The contents are mixed and the preparation is centrifuged to pelletthe buffy coat.

7. The plasma is expressed into a satellite container.

8. The residual red cells in the buffy coat can be lysed by adding ahypotonic ammonium chloride solution or other red cell lysing reagent togenerate a purified buffy coat preparation.

9. This cell preparation would be processed for PCR or other analysisusing standard methodologies. Preferably, the buffy coat is collectedwithin 24 hours from the time of donation.

Other methods can be used to obtain the buffy coat, such as the BaxterOpti®-System.

FIG. 3 illustrates schematically another embodiment of the presentinvention that can be used for purposes of validating virusdepletion/inactivation processes. The method consists of adding virus(e.g., HIV) to a blood component and then treating the blood with aviral inactivation/removal process. Of course, naturally infected bloodcan be used if desired.

Next, virus host cells are added to the blood (e.g., H9 cells for HIV orVero cells for VSV). The cells are allowed to incubate in the wholevolume of the blood component. During this incubation step, that willlast from 1 to 18 hours, infectious virus present in the blood componentattach to and penetrate the host cells to initiate the infectionprocess.

In a further step, pursuant to the present invention, the virus hostcells are harvested and separated from the blood cells by use of aleukocyte filter. The filter removes cell lines used for viralinfectivity studies. Additionally, the filter will remove and separatethe leukocytes from platelets and red cells.

The filter is then washed with tissue culture media, such as 10% FetalCalf Serum in RPMI 1640 media, as required to support the viral hostcells. The filter is then incubated at 37° C. for 6-120 hours to allow avirus to continue propagating in the host cells contained in the filter.

At the end of the incubation period, an effluent is prepared from thefilter (with or without lysing the host cells) to be used as the testmaterial. The effluent is prepared by flushing the filter with a lysisbuffer such as 1% Triton X-100 (Sigma Chemical Co.); 10 mM Tris-HCL, pH7.0; 1mM EDTA or a hypotonic solution that lysis the leukocytes but doesnot inactivate the virus.

The test material is then either tested for infectious virus usingconventional tissue culture infectivity assays (e.g., plaque-formingunits) or for viral nucleic acid markers (e.g., HIV-DNA sequences usingPCR) or viral antigens (e.g., HIV p24 antigen).

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its attendant advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

I claim:
 1. A method for providing a blood component to a recipientcomprising the steps of:removing and collecting, using a leukocytefilter, from a blood unit a majority of leukocytes present therein;using the leukocytes that have been collected in and on the filter totest the blood unit for viral contamination; and administering the bloodunit to the recipient if the test demonstrates that the blood unit doesnot include viral contamination.
 2. The method of claim 1 wherein theleukocytes are collected using a centrifugation process.
 3. The methodof claim 1 including the step of using a diagnostic assay includingnucleic acid amplification to test the blood unit.
 4. The method ofclaim 1 including the step of using a viral antigen capture assay totest the blood unit.
 5. A method for providing a blood component to arecipient comprising the steps of:removing and collecting, using aleukocyte filter, from a blood unit a majority of leukocytes presenttherein; using a nucleic acid sequencing technique to test theleukocytes that have been collected in and on the filter for viralnucleic acid sequences; and administering the blood unit to therecipient if the test demonstrates that the blood unit does not containviral contamination.
 6. The method of claim 5 wherein the leukocytes arecultured prior to being tested.
 7. A method for testing a blood unit forviral contamination without rendering the blood unit unusable fortherapeutic applications comprising the steps of:passing a bloodcomponent through a leukocyte filter; removing a majority of theleukocytes contained within the blood component; collecting a resultantfiltered blood component; creating a cell lysate from the leukocytescontained in the filter; and assaying the cell lysate for viralcontaminants.
 8. The method of claim 7 including the step of passing alysing reagent through the filter to create the cell lysate.
 9. Themethod of claim 7 including the step of flushing the filter containingthe leukocytes with isotonic saline.
 10. The method of claim 7 whereinthe cell lysate is assayed using nucleic acid amplification.
 11. Themethod of claim 7 comprising the step of culturing the leukocytes boundto the filter before creating a cell lysate.
 12. The method of claim 7including the step of using tissue culture media to culture the filter.13. The method of claim 7 wherein the filter is cultured for 1 to 5days.
 14. The method of claim 7 wherein the cell lysate is assayed usingan antigen capture assay.
 15. A method for validating a viralinactivation process comprising the steps of:treating a viralcontaminated blood component with a viral inactivation process; addingvirus host cells to the blood component; filtering the blood componentwith a filter to remove the virus host cells; and testing the virus hostcells that were captured in or on the filter for viral contaminant. 16.The method of claim 15 wherein the blood component is a naturallycontaminated blood component.
 17. The method of claim 15 including thestep of adding a virus to a blood component to make the viralcontaminated blood component.
 18. The method of claim 15 including thestep of allowing the virus host cells to incubate in the bloodcomponent.
 19. The method of claim 15 including the step of washing thefilter, after the filter step with tissue culture media, and incubatingthe filter to allow a virus to propagate in the host cells.